Modulation of luteinizing hormone and follicle-stimulating hormone in circulation by interactions between endogenous opioids and oestradiol during the ...
Modulation of luteinizing hormone and follicle-stimulating hormone in circulation by interactions between endogenous opioids and oestradiol during the peripubertal period of heifers M. W. Wolfe, M. S. Roberson, T. T. Stumpf, R. J. Kittok and J. E. Kinder Department of Animal Science, University of Nebraska, Lincoln, NE 68583, USA
The aim of this study was to determine whether the decline in oestradiol inhibition of circulating luteinizing hormone (LH) and follicle-stimulating hormone (FSH) during the peripubertal period of heifers is associated with a change in opioid modulation of LH and FSH secretion. Opioid inhibition of LH secretion was determined by response to administration of the opioid antagonist naloxone. Prepubertal heifers (403 days old) were left as intact controls, ovariectomized or ovariectomized and chronically administered oestradiol. Control heifers were used to determine time of puberty. Three weeks after ovariectomy, four doses of naloxone (0\m=.\13\p=n-\0\m=.\75 mg kg\m=-\1 body weight) or saline were administered to heifers in the treatment groups in a latin square design (one dose per day). Blood samples were collected at intervals of 10 min for 2 h before and 2 h after administration of naloxone. This procedure was repeated four times at intervals of 3 weeks during the time intact control heifers were attaining puberty. All doses of naloxone induced a similar increase in concentration of serum LH within a bleeding period. During the initial bleeding period (before puberty in control heifers), administration of naloxone induced an increase in LH concentration, but the response was greater for heifers in the ovariectomized and oestradiol treated than in the ovariectomized group. At the end of the study when control heifers had attained puberty (high concentrations of progesterone indicated corpus luteum function), only heifers in the ovariectomized and oestradiol treated group responded to naloxone. Opioid inhibition of LH appeared to decline in heifers during the time control heifers were attaining puberty. Heifers in the ovariectomized group responded to naloxone at the time of administration with an increase in FSH, but FSH did not respond to naloxone at any other time. Administration of naloxone did not alter secretion of FSH in ovariectomized heifers. These results suggest that opioid neuropeptides and oestradiol are involved in regulating circulating concentrations of LH and possibly FSH during the peripubertal period. Opioid inhibition of gonadotrophin secretion appeared to decline during the peripubertal period but was still present in ovariectomized heifers treated with oestradiol after the time when age-matched control heifers had attained puberty. We conclude that opioid inhibition is important in regulating LH and FSH in circulation in heifers during the peripubertal period. However, opioids continue to be involved in regulation of circulating concentrations of LH after puberty.
Summary.
Keywords: luteinizing hormone; follicle-stimulating hormone; opioids; oestradiol; heifer 'Current address: Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106, USA. tCurrent address: Department of Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA. JCurrent address: Department of Animal Science, University of Missouri, Columbia, MO 65211, USA.
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Introduction Maturation of the hypothalamo-hypophyseal axis during the peripubertal period is characterized by a progressive increase in secretion of luteinizing hormone (LH) in heifers (Schams et al, 1981; Day et al, 1987). Secretion of gonadotrophins is inhibited by factors originating from the ovaries before puberty. After ovariectomy, secretion of LH and follicle-stimulating hormone (FSH) is increased in prepubertal heifers, but returns to baseline values if oestradiol is administered (Schillo et al, 1982; Day et al, 1984; Wolfe et al., 1989). However, the ability of oestradiol to suppress LH and FSH secretion is reduced at puberty. Steroid feedback on gonadotrophin secretion has been postulated to be mediated in part by opioid neuropeptides. Inhibition of LH secretion by oestradiol is reversed by administering an opioid antagonist (Whisnant & Goodman, 1988). Opioids inhibit pulsatile LH secretion in prepubertal ewe lambs (Ebling et al, 1989) and opioid regulation of LH secretion does not change during sexual maturation. Decreases in sensitivity to oestradiol inhi¬ bition of LH secretion during sexual maturation are not due to changes in opioid modulation of the putative LH pulse generated in ewe lambs (Ebling et al, 1989). In contrast, opioid inhibition of LH secretion declines as female rats near puberty (Blank et al, 1979; Bhanot & Wilkinson, 1983a). We hypothesized that oestradiol inhibition of gonadotrophin secretion is mediated via opioid neuro¬ peptides in heifers and that opioid inhibition of LH secretion declines during the peripubertal period when oestradiol inhibition of gonadotrophin secretion is declining.
Materials and Methods Animals and treatments
Prepubertal heifers (\ Hereford, \ Angus, j Pinzgauer, j Red Angus) were used in the study. The 15 animals were maintained in paddocks and supplemented to allow body weight gains of 0-95 kg day-1. During collection of blood samples animals were restrained in stanchions to which they had previously been acclimatized. Feed and water were provided ad libitum throughout the sampling period.
Experimental design Gonadotrophin secretion was measured before and after various doses of the opioid antagonist naloxone were administered to ovariectomized heifers that had received a sham or oestradiol implant. The experiment was conducted during the time age-matched intact control heifers were in their peripubertal period. Thus, gonadotrophin secretion was evaluated after administration of the opioid neuropeptide antagonist naloxone, when oestradiol was inhibiting (before puberty) or no longer inhibiting (subsequent to puberty) gonadotrophin secretion. Ten heifers were bilaterally ovariectomized (403 ± 2 days of age) and half received an s.c. polydimethylsiloxane implant (3-35 mm i.d. 4-65 mm o.d. 135 mm; Dow Corning, Midland, MI) filled with oestradiol (Sigma Chemical Co., St Louis, MO) and half received a sham implant of similar size. Implants were administered caudal to the shoulder blade on the day of ovariectomy. All implants were preincubated in alcohol for 2 h and in water over¬ night. This preincubation was performed as a precaution to avoid a large release of oestradiol into the blood during the first day after implantation. Sequential blood samples were collected from the jugular vein via an indwelling cannula. Blood samples were collected from the heifers at intervals of 10 min for 4 h starting 3 weeks after ovariectomy when heifers averaged 426 days of age. This sampling frequency was used to characterize fully the response to administration of naloxone. Naloxone (Diosynth, Holland) was administered (i.v.) 2 h after the initiation of sample collection. Sampling con¬ tinued for 2 h after naloxone administration. On the basis of our preliminary studies, all changes that occur owing to a bolus administration of naloxone are detected in the first 2 h. Four doses of naloxone (013, 0-25, 0-50 and 0-75 mg kg-1 body weight) were administered to heifers in the two groups in a latin square design with one dose being administered per day. These doses were based on previous research in sheep (Malven et ai, 1984; Mathews & Murdoch, 1984). The low doses were used (lower than previous studies) to discriminate responses between different ages more clearly. Naloxone administration was repeated in heifers treated with oestradiol when heifers were 447, 468 and 488 days of age. At 516 days of age, both groups of ovariectomized heifers were administered naloxone. Gonadotrophin secretion was therefore evaluated at five different ages in heifers from the ovariectomized and oestradiol treated group. Gonadotrophin secretion was not evaluated in sham implanted heifers at the three inter¬ mediate times because a steroid-independent change in gonadotrophin secretion is not observed in heifers during the peripubertal period (Day et ai, 1984; Wolfe et ai, 1989).
All blood samples were allowed to clot at room temperature and were subsequently stored at 4°C. Blood was centrifuged within 24 h of collection at 1520 g for 15 min at 4°C. Serum was removed and frozen at 20°C until assayed. Sequential blood samples were used to evaluate mean concentrations of LH in serum and patterns of LH in circulation. Serum pools were formed for individual animals at each bleeding period by combining aliquots from all serial samples collected before (pool 1) and after (pool 2) administration of naloxone for each animal. These pools —
were
used to evaluate mean concentrations of FSH and oestradiol.
Control heifers Five heifers
(matched by age and weight with the ovariectomized heifers) remained intact to monitor time of blood samples were collected two times each week by jugular venepuncture for determination of serum progesterone concentrations. These samples were immediately placed on ice and centrifuged within 24 h. Puberty was defined as the first rise in serum progesterone greater than 1 ng ml" ' of serum for a duration of at least 12 days followed by subsequent luteal phases (rises in progesterone). puberty. Single
Hormone assays Concentrations of LH in serum were determined by radioimmunoassay (Adams et ai, 1975; Wolfe et ai, 1989) antiserum against ovine LH (TEA-RaOLH no. 35), highly purified iodinated ovine LH (LER-1056-C2) as labelled hormone, and NIH-LH-B7 as standard. Intra- and interassay coefficients of variation were 3-2 and 15%, respectively. The limit of detection for LH was 23-8 pg ml" · of serum. Antiserum made against ovine FSH (JAD-RaOFSH no. 17-6,7,9) and highly purified ovine FSH (LER-1976-A2) as labelled hormone and standard were used in the FSH radioimmunoassay (Acosta el ai, 1983; Wolfe et ai, 1989). Intra- and interassay coefficients of variation were 2-2 and 2-5%, respectively, and the limit of detection was 550 pg ml"1 of serum. Concentrations of oestradiol were determined in the pool of serum formed from serial samples collected before administration of naloxone (pool 1). All samples were analysed in a single assay that had an intraassay coefficient of variation of 1-5% and a limit of detection of 1-25 pg ml" ' of serum (Deaver et ai, 1988; Wolfe et ai, 1989). Serum progesterone concentrations were analysed for control heifers using immunoreagents obtained from Serono Diagnostics, Inc. (Norwell, MA) as validated in our laboratory (Wolfe et ai, 1989). The intra- and interassay coefficients of variation were 3-5 and 8-8%, respectively. The limit of detection was 200 pg ml"1 of serum.
using an
Statistical
analysis
Mean concentrations of LH were calculated for serial samples collected before and after administration of naloxone for each animal at each bleeding period. The deviation in mean concentration of LH and FSH subsequent to administration of naloxone was determined (mean gonadotrophin subsequent to naloxone mean gonadotrophin prior to naloxone) as well as the percentage deviation in LH and FSH after naloxone [(mean gonadotrophin sub¬ sequent to naloxone mean gonadotrophin prior to naloxone/mean gonadotrophin prior to naloxone) 100]. Response to naloxone was analysed as percentage deviation to adjust for the differences in preinjection concentrations of LH and FSH. Concentration of gonadotrophin before naloxone administration, deviation and percentage deviation in concentration of gonadotrophin were analysed using the repeated measures analysis procedure of SAS (1985). Two analyses were performed on the data. First, the effect of dose of naloxone and time were analysed in ovariectomized heifers administered oestradiol alone. The effects of dose of naloxone, time and treatment with oestradiol were then analysed using data from the first and last bleeding periods from both groups of ovariectomized heifers. —
—
Results Controls Mean onset of corpus luteum function (puberty) in control heifers occurred at 465 ± 12 days of age (mean ± sem). The average age of puberty of heifers in the control group occurred between the second and third times of naloxone administration to heifers from the ovariectomized and oestradiol treated group. Concentrations of oestradiol in ovariectomized heifers administered sham implants were below the sensitivity of the assay. Mean concentration of oestradiol in ovariectomized heifers adminis¬ tered oestradiol implants was 2-5 + 0-2pgml-1 of serum (range 2-2 to 3-2 pg ml"1). These concen¬ trations are similar to concentrations observed in intact heifers of similar age in a previous study
using the same assay system (Wolfe et al, 1989).
1. Mean concentrations of luteinizing hormone (LH) before and after administration of naloxone. Mean concentrations of LH were determined before administration of naloxone in sham-implanted, ovariectomized heifers (S) at (a) 426 and (e) 516 days of age and in ovariecto¬ mized heifers treated with oestradiol ( ) at (a) 426, (b) 447, (c) 468, (d) 488 and (e) 516 days of age. Response to administration of naloxone is depicted as a decrease (D) or increase (Kl) from pretreatment mean. Pooled standard error for pretreatment mean was 0-3 and 0-2 ng LH ml"1 of serum for the ovariectomized group at 426 and 516 days of age, respectively. Pooled stan¬ dard error for means following administration of naloxone were 0-3 and 0-3 ng LH ml"1 of serum for the ovariectomized group at 426 and 516 days of age, respectively. Pooled standard errors for the ovariectomized and oestradiol-treated group were 0-2, 0-2, 0-2, 0-4 and 0-2 ng LH ml"1 of serum for the pretreatment means at 426, 447, 468, 488 and 516 days of age, respect¬ group, ively. After administration of naloxone to the ovariectomized and oestradiol-treated i pooled standard errors for the means were 0-8, 0-6, 0-5, 0-5 and 0-3 ng LH ml" of serum at 426, 447, 468, 488 and 516 days of age, respectively, x: effect of naloxone (P < 0-01), 0-00 dose versus other doses, y: cubic effect of naloxone (P < 005). z: linear effect of naloxone
Fig.
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